C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission

Abstract

H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2 NS1217 ) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2 NS1230 and H9N2 NS1237 ). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madin-Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2 NS1230 and H9N2 NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2 NS1230 and H9N2 NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.

Using a mouse-adapted A/HK/01/68 influenza virus to analyse the impact of NS1 evolution in codons 196 and 231 on viral replication and virulence

“…Interestingly, in maHK68 the differences between the NS1(237) and NS1(230) variants were minimal, suggesting that due to adaptive mutations in the other segments the replication capacity of maHK68 is sustained and not substantially affected by the minor effects caused by this NS1 mutation. This experimental outcome is in line with the results of a similar study with a low-pathogenic avian H9N2 virus showing that elongated NS1 ORFs only slightly affected virus replication and IFN induction [44]. However, the robust replication capacity of maHK68 permitted the characterization of the NS1 variants in the context of an inactivated RNA-binding domain caused by the R38A exchange.…”

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens

“…13 In our study, compared to the Hp virus, the NS1 mutants exhibited impaired virus excretion from infected birds at 4dpi particularly via the cloacal route. A reduced amount of virus excretion due to NS mutations may reduce bird-to-bird transmission 26 which, however, was not studied in our experiment. Moreover, spread of influenza viruses to the brain usually occurs by the hematogenic route and also through the olfactory nerve.…”

Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus

“…More recent serological evidence suggests that dogs have been naturally infected with human seasonal H1N1 and H3N2 viruses (28), avian H5N1 (29), H5N2 (30), H9N2 (31), and H10N8 (32) viruses, and the 2009 H1N1 pandemic virus (pdmH1N1) (33). In China, more than 20% of domestic dogs were shown to have serum antibodies to pdmH1N1, and dogs positive for antibodies to both CIV-H3N2 and pdmH1N1 have been identified (23,34). During a surveillance effort in 2012, researchers identified a virus isolate that contained the hemagglutinin gene segment from CIV-H3N2 and the remaining 7 gene segments from pdmH1N1 (35); more recently, another isolate that contained the matrix gene segment from pdmH1N1 and all other gene segments from CIV-H3N2 was identified (36).…”